對目標細胞具有專一結合力的生物標誌在診斷和標靶治療中是非常重要的工具。篩選對目標細胞表面具有專一性和親和力的生物標誌的傳統方法有一些缺點，包括耗時、費力、需要經過訓練的操作人員、需要大型且昂貴的實驗設備並且需要消耗大量的試劑，本研究利用微流體平台進行生物標誌篩選，因為其具有可以在較短的時間內自動化完成篩選生物標誌的能力。噬菌體呈現胜肽庫作為篩選對細胞表面具專一性的胜肽是一個有深具潛力的工具。近幾年來人們對於一部份的癌細胞被認為是癌症幹細胞，或是稱做腫瘤啟動細胞有極大的興趣。這些細胞被認為具有使一般細胞分化為癌細胞的能力，也是造成癌轉移的主要原因。可以分辨癌細胞和癌症幹細胞的專一性胜肽對於癌症的早期診斷和標把治療是一個非常重要的工具。在本研究中，我們利用整合型微流體系統成功地完成了M13噬菌體呈現胜肽庫的篩選實驗，其中目標細胞則選擇用台灣最常見的癌症之一的大腸、結腸癌。我們在噬菌體呈現胜肽庫篩選實驗中加入了負向篩選，相較於傳統篩選實驗步驟只有正向篩選且需要將近一個月，微流體系統可以在更少的樣本和試劑用量之下，自動化的使用單晶片並在36小時內完成整個篩選實驗。而且微流體系統在本實驗中被證明有更好的細胞專一性，在篩選流程完成後，針對大腸癌篩選出3個胜肽生物標誌，而針對大腸癌幹細胞找出4個胜肽生物標誌。這個新發展的技術，對於癌症的早期診斷和癌轉移病人的標把治療將會極有幫助。

Affinity reagents that binding to specific targets are essential tools for clinical diagnostics and targeted therapeutics. However, benchtop methods for probing for target molecules with affinity reagents have some several drawbacks; they are , time-consuming, labor-intensive, and require of large, typically expensive instruments that require both expensive reagents and large sample quantities. Microfluidic platforms could potentially automate the screening process within a shorter period of time. Phage display peptide library libraries are promising tools for in selection of cell surface peptides and could be used for this purpose. In recent years, a subpopulation of tumor cells are considered as cancer stem cells, or as tumor initiation cells which are of great interest. The subpopulation of cells is believed to have an ability to initiate the differentiation of cells to cancer cells, and they are the main cause of metastesis. A specific peptide that can recognize and differentiate cancer cells and cancer stem cells is extremely useful in early diagnosis and target therapeutics. In this study, selection of M13 phage displayed peptides that bind to colon cancer cells and colon cancer stem cells using an integrated microfluidic system was successfully demonstrated. In addition to positive selection process, we provided a negative selection into the screening process of phage display library. Compared with the traditional method (which needs at least one month), the total selection process was significantly shortened to 36 hours. In this study, 3 candidates for colon cancer cell and 4 candidates for colon cancer stem cells after the total selection process were successfully screened. The selected peptides were tested to show relatively high specificity on target cells. The developed technique may be promsing for early diagnosis of cancer and target therapeutics for metastasis cancer patients.

摘要 III
Table of contents IV
List of figures VI
List of tables X
Abbreviations XI
Nomenclature.......................................................................................XIII

Chapter 1 Introduction 1
1.1 Colon cancer cells (CRCs) and colon cancer stem Cells (CSCs) 1
1.2 Isolation of CSCs 4
1.3 Phage display library 6
1.4 MEMS and Bio-MEMS 8
1.5 Motivation and objectives 11

Chapter 2 Design and fabrication 15
2.1 Integrated microfluidic chip of phage display 15
2.2 Fabrication process 17

Chapter 3 Materials and methods 23
3.1 Preparation of the experimental materials 23
3.1.1 Preparation of CRCs and Colon CSCs 23
3.1.2 Phage display library and E. coli K12 ER2738 23
3.1.3 Preparation of PCR reagents 24
3.1.4 Preperation of specific peptide coated magnetic beads 25
3.2 Experimental process 27
3.3 Characterization of the microfluidic sytem 29
3.3.1 Characterization of the microfluidic system: pumping rate 29
3.3.2 Characterization of the microfluidic system: shear force 30
3.4 Specificity assay 31
3.5 Kd value assay 32

Chapter 4 Results and discussion 36
4.1 Characterization of the integrated microfluidic system 36
4.2 On-chip culture of E. coli and cancer cell line 39
4.3 Screening of cell specific oligopeptide 40
4.4 Sequencing and bioinformatics assay of the specific oligopeptide 41
4.5 Specific assay of the HCT-8 and CSC specific peptide 42
4.6 Measurement of Kd value 43
4.7 Comparison of the traditional phage display method and the microfluidic system 44

Chapter 5 Conclusions and future perspectives 57
5.1 Conclusions 57
5.2 Future perspectives 58

References 59

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